Colorectal cancer (CRC) is the third most diagnosed and deadly cancer in the United States. Numerous epidemiological studies have shown that a high-fiber diet is correlated with decreased incidence of CRC. Although the underlying mechanism is unknown, we hypothesize that dietary fiber has a protective effect because it is metabolized by anaerobic bacteria in the colon and fermented into butyrate. Butyrate is a potent histone deacetylase (HDAC) inhibitor that alters epigenetic events and gene expression and might maintain cell proliferation and cell death homeostasis to prevent CRC. The objective of this application is to investigate the mechanistic link between dietary fiber, gut microflora, butyrate, and epigenetic modifications which result in gene expression changes that alter cancer susceptibility. To tightly control experimental variables, the proposed experimental plan utilizes several mouse models of CRC. ApcMin/+, azoxymethane (AOM) treated, and AOM plus dextran sodium sulfate (DSS) treated mice will be maintained with defined gut microflora or germfree and be fed high- or low-fiber diets to determine whether gut microflora is required for a high-fiber diet to protect against CRC. To determine whether bacterial fermentation of fiber into butyrate is the mechanism of the fiber effect, germfree mice will be recolonized with bacteria that produce high or low levels of butyrate. CpG methylation and gene expression will be analyzed in a genome-wide manner using methylated DNA immunoprecipitation (MeDIP) combined with BAG and CpG island microarrays and transcript profiling. CpG methylation and gene expression profiles from mice fed a high- or low-fiber diet in the presence or absence of gut microflora will be compared. Superimposing these data sets will identify CpGs with altered methylation that are also associated with changes in gene expression and cancer susceptibility. These sites will be considered candidates for contributing to altered risk for CRC. To understand the role of dietary fiber and gut microflora at a molecular level, epigenetic events will be analyzed at several candidate genes before and after the onset of tumor formation using chromatin immunoprecipitation (ChIP) assays, sodium bisulfite mutagenesis followed by sequencing, and DNase I hypersensitivity assays. This work should elucidate the mechanisim of the fiber effect on CRC susceptibility and may lead to new preventative strategies for CRC.